Application of Fluorescence Microscopy to a Study of Chemical Problems R. S. Davidson Department of Chemistry University of Kent Canterbury UK CT2 7NH 1 Introduction This review aims to show the reader how the use of fluorescence microscopy using a conventional or modified fluorescence micro- scope may be used to study chemical problems such as the photo- degradation of naturally occurring polymers photoinduced reactions the dyeing of fibres and the measurement of Tg (glass transition temperature) values. Hitherto the use of fluorescence microscopy has been the domain of the biologist but now there are opportunities available to the chemist. Fluorescence may be defined as light which is emitted when an electronically excited state relaxes to an electronic state of lower energy and possessing the same spin state. For organic species in solution and in the solid state the fluoresence observed is usually associated with relaxation from the first excited singlet state to the ground state.' Needless to say this is only one of the ways that an electronically excited singlet state can relax and these processes are usually shown pictorially by means of a Jablonski diagram (Fig. 1). A wide range of compounds exhibit fluorescence. One of the features that marks highly fluorescent compounds is that they possess rigid structures. Examples include polycyclic aromatic hydrocarbons (e.g.anthracene) and dyes2 which are based on a fused aromatic ring system (e.g.fluorescein 1 the coumarin derivative 2 Fig. 2). The rigid structure prevents the electronically excited state de- activating via undergoing intramolecular isomerisation (e.g. as exemplified by stilbenes which undergo cis-trans isomerisation3) or conformational changes (e.g. twisting about the 1-1' C-C bond in 1 ,l'-binaphthyl). In some cases intramolecular motion can lead to a new emitting species which does not possess an equivalent R.S. Davidson gained an ARICfrom Leeds College of Technology in hv -hip (fluorescence) -hv (phosphorescence) or A or a 'I I Figure 1 Jablonski diagram (A. Jablonski Nature 1933 131 839; Z. Phys. 1935,94,38). Fluorescein 1 A coumarin laser dye 2 Figure 2 Some fluorescent dyes. by bond rotation1958 and then curried out research for a PhD degree under the supervision of Professor B. Lythgoe FRS at the University of Leeds. This work led to a successfil synthesis of tachysterol and the award of the J. B. Cohen prize from the University of Leeds. Having spent a year working with Professor R. B. Woodward (Harvard University) on a synthesis of vitamin B he took up the post of lecturer in organic chemistry at the University of Leicester (I964)where he pursued his interests in photochemistry. Particular interests at this time were photoinduced electron-transfer reactions and photo-oxidation reac- tions. He was awarded a DSc degree from the University of Leeds in 1978. He moved to the City University London in 1979 to take up the chair of organic chemistry. It wus whilst in this post that his inter- est in microscopy and the photodegradation of natural polymers was awakened. In 1990 he moved to the University of Kent where he is currently Professor of Applied Chemistry. Most of his research is concerned with radiation curing (photoinitiated poly-merisation processes) and he is also investigating ways to prevent the photoyellowing of papers made from high yield pulps. He had the title of Emeritus Professor of Organic Chemistry conferred upon him by City University in 1993. He has over 200 publications in the field of photochemistry. 3 Twisted S state of 1 ,l-binaphthyl Almost planar relaxed S state of 1 .I-binaphthyl Figure 3 Relaxation of the initially created excited singlet state of 1 ,l'-binaphthyl by bond rotation. (MFM Post J. Langelaar and J. D. Van Voorst Chem. Phys. Lett. 1975,32,59). stable ground state i.e. emission occurs from the almost planar form of 1 ,I'-binaphthyl 3 (Fig. 3). As a consequence the fluorescence spectrum of 1,l '-binaphthyl shows a broad structureless band at lower energy than that expected from the primarily excited species i.e. the intramolecular motion has generated a sizeable Stokes shift. Compounds which exhibit such shifts and possess high quantum yields of fluorescence are par- ticularly valuable as fluorescence probes4 and no more so than when the probes are used in fluorescence microscopy. Many compounds exhibit fluorescence in solution which does not emanate from the initially created excited singlet state but rather may be attributed to the excited state undergoing solvation (thereby giving rise to solva- tochromic shifts) deprotonation or protonation intramolecular charge transfer etc. If the fluorescence spectrum of a compound is sensitive to solvent polarity then such a material may be used to probe the polarity of cell membranes etc.5 and such a process can be visualised by means of fluorescence microscopy. The phenome- non of excited singlet states undergoing protonation and deprotona- tion is well established6 and is well illustrated by the hydroxy coumarin 4' (Fig. 4). 24 1 HO Figure 4 Some protonation and deprotonation reactions exhibited by 4 Fluorescent species which are sensitive to pH have found application in determining the pH of intracellular material * A topic which is currently attracting much attention is that of intramolecular charge transfer and fluorescence spectroscopy has been used exten- sively to study the dynamics of the process Perhaps the most fre- quently studied compound which exhibits this phenomenon is 4-dimethylaminobenzonitrile This compound exhibits a broad structureless emission band which shifts to lower energy and exhibits a decrease in quantum yield as the solvent polarity increased Such behaviour is the hallmark of an excited state which possesses a considerable amount of charge transfer and conse- quently has a high dipole moment Many experimental results support the view that excitation of the aminobenzonitrile leads to rotation about the C-N single bond with concomitant electron transfer from the amino to the cyanobenzene moiety (Fig 5)thereby leading to a twisted intramolecular charge transfer (TICT) Figure 5 Formation of a twisted intramolecular charge transfer complex Many compounds have been found to behave in a similar fashion to the aminobenzonitrile and compounds of particular value in fluo-resence analysis (eg end group determination of peptides using the Edman degradation)lO and fluorescence microscopy are sulfonated amino naphthalenes such as 1-dimethylaminonaphthalene-5-sul-fonic acid4 Since the formation of a TICT complex involves a change in molecular conformation it is not surprising to find that the efficiency of the process is dependent upon solvent viscosity Thus such compounds have found use as probes for monitoring the change in viscosity which occurs when materials such as acrylates undergo polymerisation I The occurrence of charge transfer in excited states is not limited to examples where the donor is directly linked to the acceptor group Thus many examples are known of compounds which exhibit excited state charge transfer in which the donor and acceptor groups are separated by a rigid spacer group or a flexible chain '2 Compounds having a donor and acceptor group separated by rigid spacers (eg 5 Fig 6) of varying dimensions have been used to show that electron transfer can occur over large distances eg 25 A For compounds having donor and acceptor CH CN CH 5 Figure 6 A rigid compound which exhibits long distance electron transfer (N Paddon Row and J W Verhoeven New J Chem ,1991,15 107,J W Verhoeven Pure Appl Chem 1990,62,1585 CHEMICAL SOCIETY REVIEWS 1996 OH groups linked by a flexible chain the efficiency of excited complex formation can in part or wholly be dominated by the conformational flexibility of the chain For compounds such as 6 (Fig 7) it is 6 Figure 7 Formation of an intramolecular excited charge transfer complex necessary for the donor group (the amine) to interact with the T-electron system of the aromatic hydrocarbon acceptor group l2 Another requirement for compounds such as 7 to exhibit excited charge transfer complex formation is that the amino group is able to donate an electron This is cleaily impossible when the amino group is protonated or if the lone pair of electrons is part of a dative bond This simple fact has been used to design compounds which will act as molecular switches and signalling devices Compound 7 (Fig 8) 7 Intrarnoleculs?r excited state charge transfer complex Intramolecular excirrer formation Figure 8 A compound which exhibits both excimer and exciplex formation exhibits intramolecular excited charge transfer complex formation in neutral solution eg ethanol but in the presence of acid the formation of an excited charge transfer process is switched off and is replaced by intramolecular excimer formation I3 Since the two types of complex emit at different wavelengths they can be readily distinguished Compounds have been designed which exhibit intra- molecular excited charge transfer complex formation and which have been used as molecular switches in which the on-off process is regulated by pH l4 In many cases intramolecular electron transfer leads to fluorescence quenching and consequently if in these com- pounds the donor ISan amino group protonation or involvement of APPLICATION OF FLUORESCENCE MICROSCOPY TO A STUDY OF CHEMICAL PROBLEMS-R S DAVIDSON 8 9 Figure 9 Compounds which exhibit an increase in fluorescence yield when the lone pair electrons on nitrogen are involved in bonding the nitrogen lone pair in complexation leads to enhancement of the fluorescence quantum yield Protonation of the nitrogen atom in 8 (Fig 9) leads to a spectacular increase in quantum yieldls and in the case of compound 9 (Fig 9) complexation with potassium led to a 47-fold increase in fluorescence intensity I6 A process which is somewhat similar to quenching of fluo- rescence by intramolecular complex formation is that of quench- ing via the heavy atom effect Quenching by halogen atoms increases in efficiency as the atomic mass of the halogen atom is increased and consequently bromine is a more efficient quencher than chlonne For quenching to be observed the halogen atom may be directly attached to the fluorogenic chromophore or it may be linked to the chromophore via a flexible chainI8 or alternatively may be present in solution as part of the solvent Where the atom is present in the molecule its removal via ground- or excited-state reactions will lead to an increase in fluorescence yield An example which illustrates such a process is afforded by reactive dyes 10 11 and 12 (Fig 10) These dyes become covalently attached to wool via nucleophilic displacement of a halogen group by the €-amino group of lysyl groups present in the wool It was found that this process in itself did not remove all the halogen atoms and that further reaction with external reagents such as water or an amine was necessary for the dyes to exhibit their maximum fluorescence intensity l9 L1 10 2 Fluorescence Spectrometers and Microscopes For the routine recording of fluorescence spectra a spectrometer which contains the basic elements shown in Fig 11 can be used It is a relatively simple task to replace the sample accessory (3) by one which will accommodate solid samples 20 In order to decrease the influence of scattered light upon the emission spectrum it is best to avoid the right angle configuration and to have a system whereby the angle of the sample with respect to the excitation beam can be varied In this way the angle can be varied so as to find the one which gives the least disturbed and highest intensity signal With a spectrometer such as the one shown in Fig 11 the monochromators are equipped with entrance and exit slits and by appropriate choice of slit widths maximum spectral resolution can be achieved If the fluorescence spectrum of the sample does not exhibit a Stokes shift t Ia Figure 11 Layout of the components of a spectrofluorimeter (1) Light source usually Xe or Hg/Xe arc lamp (2) Excitation monochromator (3) Sample shown as a quartz cuvette in which the emission is observed at right angles to the exciting beam (4) Emission monochromator (5)Photomultiplier tube (6) Amplifier (7) Data recording device great care has to be taken to ensure that scattered exciting light does not affect the spectrum and that the emission spectrum is not per turbed by some of the fluorescence being absorbed by the sample (inner filter effect) When weakly emitting samples are being exam- ined better sensitivity can be achieved by operating the detection system in the photon counting mode 21 With such a spectrometer it is possible to use the exciting light as a means of bringing about photochemical reactions in the sample and if those reactions cause a change in fluorescence intensity or spectral shift the course of the reaction can in principle be monitored in real time This can be dif- ficult to achieve in practice since usually it is necessary to employ large slit widths for the excitation monochromator which can lead to difficulties in recording the spectra However it is a relatively simple job to record spectra after set illumination times by chang- ing the slit widths In this way the photodimerisation of styrylpyridinium groups (Fig 12)appended to a poly(viny1 alcohol) (PVA) was monitored 22 The spectra in Fig 13 show that the excimer emission exhibited by the films of modified PVA decreases 11 $03 *a IBr 'S03Na IBr Figure 10 Reactive dyes which are non fluorescent but which become flu0 rescent upon removal of the halogen atoms via nucleophilic displacement Figure 12 The photodimerisation of a styrylpyridinium salt I4O I 360 380 400 420 440 460 480 hInm Figure 13 Fluorescence spectra recorded during irradiation of styrylpyridin-ium groups pendant to a poly(viny1 alcohol) chain. in intensity as reaction proceeds. At the end of the reaction some styrylpyridinium groups remain and these are presumably groups which do not have a neighbour that is sufficiently close to enable the chemical reaction to occur. Many of the benefits which accrue from using a fluorimeter of the type shown in Fig. 11 are due to the presence of the two monochromators. If these are replaced by filters the recording of spectra becomes impossible and for emission to be observed it requires that the excitation and observation wavelengths are suffi- ciently well separated. The conventional fluorescence micro- scope23 relies upon the use of filters and consequently fluorescence probes etc. being examined via the microscope should exhibit large Stokes shifts. The examples cited (i.e.com-pounds 1-12) possess this property and many well utilised probes exhibit fluorescence properties which are due to the photophysical processes displayed by compounds 1-12. Microscopes such as the one shown in Fig. 14 are the standard work-horse of immunology laboratories where fluorescent labels are used to detect particular interactions. In order to obtain sufficiently high light levels for visual observation a super-high-pressure mercury lamp is the usual source of excitation. A filter is used on the excitation side which either transmits a wavelength associated with one of the main emission lines of the lamp e.g. 365 nm or transmits a spectrally wide band of light. The dichroic mirror (Fig. 15) reflects the excitation light onto the sample but transmits the fluorescence pro- duced by the sample. With the advent of charge coupled device (CCD) cameras it is possible to observe very low light intensities and such cameras can be mounted onto the microscope so as to increase the sensitivity of fluorescence detection. When the microscope is being used for studying chemical reactions it is often advantageous to mount either a photomultiplier tube or a monochromator equipped with a photomultiplier tube on the microscope. In this way fluorescence CHEMICAL SOCIETY REVIEWS 1996 0Viewing Optics I i----Optional filter! I iSample Figure 14 Diagram showing the parts of a fluorescence microscope associ- ated with revealing the fluorescence of the sample. intensity changes during irradiation can be recorded and if the monochromator is properly equipped fluorescence spectra can be recorded. Such a system can be enhanced further if a continuous wave (cw) e.g. an argon ion or helium cadmium laser replaces the lamp. By placing a timed shutter between the laser and the micro- scope the system can in principle be used for determining fluo- rescence lifetimes (provided the photomultiplier tube is operating in the single photon counting mode) and to carry out bleach recovery experiments (see later). The layout of such a system is shown in Fig. 16. Such an experimental set-up is ideal for examining samples and for carrying out photochemical reactions. The high intensity excitation beam can be used simultaneously to bring about a photochemical reaction and also monitor fluorescence changes which occur during the reaction (real time fluorescence spec- troscopy). However if the sample requires heating or if the sample is to be reacted in solution there is insufficient room between the microscope objective and the sample to allow positioning of the necessary equipment. This problem can be overcome by the use of an inverted fluorescence microscope. In such a microscope all the optical parts are sited below the microscope stage thereby leaving space between the stage and the ceiling of the room in which the microscope is being used to house any equipment. For purposes outlined later we have constructed two accessories which fit on the top of the stage and which allow polymer films and similar samples to be heatedz4 and another in which materials can be sub- jected to chemical treatment.25 These accessories are shown in Fig. 17. A very useful commercially available accessory is a micro- injector which enables metered amounts of material (e.g. a dye) to be injected into a sample being examined on the stage of the micro- scope. iilnrn i./nrn Mirror for visible light excitation. Mirror for UV light excitation. Figure 15 Spectral characteristics of a dichroic mirror used in a fluorescence microscope. APPLICATION OF FLUORESCENCE MICROSCOPY TO A STUDY OF CHEMICAL PROBLEMS-R S DAVIDSON P-Computer -Oigitiserx-Amplifier Photomultipliertube L Shutters -Pockel cell ns ’ -Acoustooptical AlA-I. t. /Sample open shut A (ms) Figure 16 Diagram of a fluorescence microscope adapted for recording spectra fluorescence lifetimes and for carrying out bleach recovery experiments (a) Copper_ blocks Peltier pump Copper block .. Hole for Insulation sample holder Top face Slide UV cured polymer -Dye crystals I \Irradiated (bottom face hv Figure 17 Accessones for use with an inverted fluorescence microscope (a) for heating a polymer film (b) for carrying out chemical reactions 3 Some Examples of the Use of Fluorescence Microscopy to Study Chemical Problems 3.1 The Chemistry of Wool The structure of a wool fibre is very complex and Fig 18 shows in diagrammatic form some of its components The cuticle is normally only one cell thick except where the cells overlap and is rich in cystine Whilst most of the fibril structure is made up from keratin (a protein) molecules the cortical cells are separated from each other by a cell membrane complex which is largely composed of lipids The cell membrane complex also separates the cuticle cells from the underlying cortical cells Whilst much of the chemistry of wool is based on the fact that it is a proteinaceous fibre it is clear that this is very much an oversimplification of the real case Like other natural polymers wool is affected by light The most obvious effect is that of photoyellowing caused by wavelengths <380 nm photobleaching is caused by wavelengths >380 nm The photo- degradation processes also lead to an increase in the fluorescence of wool fibres26 (Fig 19) Clearly the colour changes indicate that photochemical reactions proteln molecule posslbly comprlslng three polypeptide chains helices) twistedlogether hellcat rrrmgement of proteln molecules nuclear remnant 1 mlcrollbrll p.r.cortex mrcrollbrll orthoconexcotllcalCI Figure 18 Sketch of a broken section of fine wool fibre showing the major cellular components and the detailed structures within them are occurring but in addition to these others occur which lead to the wool fibres losing their strength These changes are exacerbated when the wool has been subjected to treatments such as oxidative bleaching or the application of fluorescent whitening agents (FWAs) It has been known for some time that wool possesses an intrinsic fluorescence the origin of which is far from understood When sections of wool fibres are examined by fluorescence microscopy it was very evident that the tips were far more fluo- rescent than the roots 26 By measuring the fluorescence intensity along the length of a 60 mm fibre (obtained from Merino sheep) it was found that most of the fluorescence was exhibited in the first 5 mm from the tip of the fibre 27 Given that the fleece is densely packed this result is not surprising When a fibre from a more open- structured fleece was used the difference in fluorescence intensity at the root and tip was not so marked 28 Fluorescence spectra of the tips and roots of Merino wool (obtained by microspectrofluorim- etry) were found to be very similar which suggested that the same species are present in tips and roots which give rise to the fluo- rescence A further finding which substantiated this deduction is that the bleach-recovery profiles for the tips and roots are similar In this type of experiment the sample is irradiated for a short time CHEMICAL SOCIETY REVIEWS 19% Figure 19 Wool fibres before irradiation (left hand side) and after irradiation (right hand side). (e.g. 1 ps),the intensity of fluorescence measured and then the fluo- rescence intensity measured after the sample has remained in the dark some time e.g. 2 ms. If the fluorescent species undergoes reduction to give a leuco species oxidation of this species in the dark period will regenerate the fluorescence. In other cases recov- ery of the fluorescence can be due to fluorescent species migrating into the viewing area of the microscope during the dark period. Such an experiment carried out with the tips and roots of a fibre showed that the fluorescent species was destroyed upon irradiation and that the destruction occurred over a similar time period for tips and roots. The origin of the fluorescence of wool has been the subject of much debate. Conventional fluorimetry has been used to show that several species are responsible for fluorescence. When the wool is excited at 300 nm most of the emission (Amax 350 nm) emanates from tryptophan. Excitation with light of wavelength >300 nm generates fluorescence having maximal intensity at >350 nm; e.g. excitation at 375 nm generates fluorescence having a A, at 430 nm. Species which may be responsible for this long wavelength emission include carbolines and other compounds which are derived by degradation of tryptophan. It is clear from the bleach- recovery experiments that these compounds are destroyed to non- fluorescent products upon irradiation and this is clearly the origin of the photobleaching effect. The destruction process can be accom- plished chemically under both oxidising and reducing conditions which suggests that there is not a unique photodestruction process. It has also been established that the fluorescence of these com- pounds is quenched by the disulfide bonds present in cystine and this phenomenon may contribute to the lack of fluorescence exhib- ited by the root of the fibre (where little photooxidation of cystine has occurred) and the much greater fluorescence intensity of the tips (where much of the cystine and tryptophan has been oxidised26). The dyeing of wool usually utilises the fact that it contains free amino groups (the €-amino groups of lysine) and to a lesser extent sulfhydryl groups (present in cysteine and can be chemically pro- duced by reduction of cystine). The presence of the amino groups means that anionic dyes (usually containing sulfonic acid groups) and reactive dyes (which rely upon the amino group acting as a nucleophile and thereby forming a covalent bond with the dye viu a Michael addition reaction and in other cases via a nucleophilic sub- stitution reaction). Since the wool fibres are more open at the tips due to photodegradation etc.,dyeing occurs preferentially at the tips thereby leading to uneven dyeing. This problem can be overcome by the use of levelling agents which aid migration of the dye within the fibre. In an alternative approach to obtaining level dyeing wool has been treated with chitosan (an amino-polysaccharide) .29 It was expected that the chitosan would adhere to the surface of the wool fibres and that the presence of the amino group would lead to rapid adsorption of anionic dyes. Having obtained a high concentration of dye on the surface the normal processes whereby the dye is taken into the fibre were expected to take over. Fluorescence microscopy was used to demonstrate that chitosan-treated wool fibres when treated with a fluorescent whitening agent underwent rapid dyeing at a lower temperature and that most of the dye was located on the exterior of the fibre. By raising the temperature of the dye-bath the dye on the surface of the fibre migrated into the interior of the fibre leading to a greater degree of level dyeing than was observed with wool that had not been treated with chitosan. By construction of the appropriate accessory (Fig. 17) and using it with an inverted microscope it proved possible to follow the dyeing of wool in situ.I9 To follow the dyeing of a fibre in situ it is essential that the dye-bath solution does not fluoresce (or at least is only weakly fluorescent) so that the fluorescence of the fibre can be readily detected by the dye or if the intensity of fluorescence is being monitored that the detection device is only seeing fluo- rescence from the fibre. This requirement was accommodated by the dyes 12 (Fig. 10)and N-(9-acridinyl)maleimide. Both dyes react with primary amines and sulfhydryl groups but selectivity can be obtained by control of pH. Nucleophilic attack upon the bromo- acrylamido group present in 12 leads to loss of bromine thereby removing the internal quenching group and rendering the stilbene dye fluorescent. The maleimido group present in N-(9-acridiny1)maleimide quenches the fluorescence of the acridinyl group and consequently where the maleimido group is converted to a succinimido group via nucleophilic attack of an amino or sulfhydryl group the fluorescence of the acridinyl group is restored. The dyeing of wool fibres by these dyes was carried out on the microscope stage and the process of the dyeing followed by record- ing the change in fluorescence intensity of the fibres with time. Figs. 20 and 2 1 show the results obtained in this way and Figs. 22 and 23 show photographs of the wool fibres before and after dyeing. Figs. 20 and 21 demonstrate that the dye-bath solution exhibits little fluorescence that the wool fibres exhibit an increasing amount APPLICATION OF FLUORESCENCE MICROSCOPY TO A STUDY OF CHEMICAL PROBLEMS-R.S. DAVIDSON 140 250 120 c.-C3 $ 100 C3 v200 .-E *O vl a,w .S 60 8 c ' 40 rr 150 9 Tipfibre 12 A Middle fibre 1G 20 V Tipfibre2+ Middle fibre 2 0 Background0 I I I I I I 100 0 1 2 3 4 5 6 tlh Figure 21 Change in fluorescence intensity of wool fibre present in a dye- bath solution containing N-(Pacridinyl) malemide as a function of time. of fluorescence as their immersion time in the dye-bath increases 50 and that the cut end of the fibres takes up the dye more rapidly than the centre portion of the fibre. This latter observation can be attrib- uted to some of the dye entering the fibre via the exposed centre of Solution the fibre rather than through the cuticular layer. Such experiments Ppave the way for a more detailed investigation of how different parameters e.g. varying the amount and consistency of dye-bathI I I I I 1 2 3 4 5 agents such as surfactants and levellers affect the dyeing process. tlh In order to achieve the whiteness required by customers fluo-rescent whitening agents (FWAs) are applied to wool. These colour- Figure 20 Change in fluorescence intensity of a fibre present in a dye-bath less dyes absorb ultraviolet radiation and emit blue light thereby containing 12 as a function of time. making up for the blue light which is absorbed by the yellow coloured species which are responsible for the wool having an off-white colour. Unfortunately FWAs photodegrade to give yellow products via singlet oxygen and radical mediated reactions and in Figure 22 Wool fibres before dyeing. Figure 23 Wool fibres after dyeing with 12 for 6 h at 80 "C. the process increase the rate of yellowing of the wool. This process can be readily monitored using a fluorescence microscope. Fig. 24 shows that wool fibres treated with an FWA exhibit little fluo- rescence after they have been exposed to light. The degradation of FWAs on the surface of wool has been mon- itored in real time using fluorescence microscopy. Wool was dyed with FWAs based on the stilbene and pyrazoline chromophores and then irradiated on the stage of a fluorescence microscope with simultaneous recording of the fluorescence intensity of the sample.30 Using this technique the ability of additives such as CHEMICAL SOCIETY REVIEWS 1996 Blankit D (80%formaldehyde sulfoxylate) and thiourea dioxide to arrest the degradation of the FWAs was investigated. A diagram-matic representation of one of the many results is shown in Fig. 25. It was found that the degradation of the FWAs was preceded by an induction period which was attributed to the wool protecting the FWA by reaction of its cystyl residues with any generated singlet oxygen. However the protective action of the cystyl residues is sacrificial by nature since in the process it becomes oxidised with the final product being cysteic acid. Once the cystyl residues in the wool (particularly the cystyl-rich cuticle) have been consumed degradation of the FWAs commences. The role of singlet oxygen is not as great for the stilbenes as it is for the pyra- Figure 24 Wool fibres treated with an FWA before irradiation (left hand side) and after irradiation (right hand side) APPLICATION OF FLUORESCENCE MICROSCOPY TO A STUDY OF CHEMICAL PROBLEMS-R S DAVIDSON Tirne/mins Figure 25 Degradation of an FWA as monitored by real time fluorescence spectroscopy Real time degradation of pyrazoline H treated wool in the presence and absence of a 2% solution of thiourea dioxide where r IS the time for the fluorescence intensity to decrease by lo%,rso is the time for the fluorescence intensity to decrease by 50% and r4' = r -I, zolines and there is evidence that a significant part of the degrada tion of the stilbenes involves a reductive interaction between the wool and the FWA 3.2 The Photobleaching and Photoyellowing of Paper Containing Lignin The mechanical strength of plant materials is due to the laying down of a polymer Iignin in the cell walls Lignin is derived from phenyl tyo" co :t120H I -YH CH2W 6 alanine via a series of enzymatically induced hydroxylation and oxidation reactions It does not have a unique structure and the chemical structure of an extracted lignin will reflect the nature of the species from which the lignin was extracted and the season in which the lignin was laid down Despite the complexity of the material several important structural motifs have been identified (Fig 26) and many of these contain photoactive chromophores such as the a-0-4 and p-0-4 units quinones and phenolic residues If pulp produced from wood without extracting the lignin is converted into paper the product has a light brown appearance Bleaching of the pulp can be used to produce papers having an acceptable white ness but such papers readily undergo photoyellowing As with wool the yellowing is largely produced by light wavelengths <396 nm with longer wavelength light leading to photobleaching There IS abundant evidence to support the view that the lignin is to a large extent responsible for the photoyellowing Extensive detailed work has unravelled many of the processes which lead to the formation of coloured products but as yet little precise information is available as to the structure of the degradation products 31 Lignin is ff uorescent and consequently the structure of a sample of wood can be examined by fluorescence microscopy By use of the chemical reactor shown in Fig 17 the in situ delignification of wood using a Kraft liquor at 95-100 "C was continuously moni tored The loss of lignin was readily apparent and after a 4 h treat-ment some lignin still remained attached to the cellulosic t12COH tI p r II tIC -co I CHOtl CII3 I I cu,o 0-HC -ftl H,CO 4 b -'HFoH I I 0-$H OCH OH C-0 I OH ti a phenylcoumarin unit fphenolic OH group b a 0 4 linkage g methoxy group c p 0 4 linkage h p 1 linkage -0d 5 5' bond biphenyl unit i methylene quinone bond -0 e pinoresinol unit Figure 26 Structure of lignin after Freudenberg (C K Freudenberg and A C Neish Mol Bra1 Biochem Biophysrcs 1968,2 103) -framework The fluorescence of this lignin was different in appear-ance to that observed prior to the Kraft liquor treatment which would indicate that it has a different structure Whether this struc- ture has been produced by the chemical treatment or not is not clear The presence of lignin in the wood structure can also be detected by staining with dyes such as Basocryl gold and Astrazon red Since lignin is fluorescent it is not surprising to find that papers made from high yield pulps i e pulps produced without delignifying the wood also exhibit fluorescence 32 The wavelength distribution of the fluorescence is highly dependent upon the excitation wave- length that is used and this shows that more than one chromophore is responsible for fluorescence When such papers were subjected to bleach-recovery experiments (using the modified fluorescence microscope) with an argon ion laser operating at 488 nm as the illumination source it was found that the recovery was relatively slow (>2 s) This is attributed to lignin diffusing into the area where the lignin had previously been photodegraded and is an indication of the lignin being a relatively mobile species Chemical and photo- chemical reduction of papers made from high yield pulps increased the overall fluorescence intensity of the papers and fluorescence spectra showed that this was due principally to species emitting at 400 nm It is possible that the reduction process has transformed species which act as inner filters or quenchers of fluorescence into innocuous products e g quinones into dihydroxybenzenes coniferaldehyde into coniferyl alcohol These changes are unfortu- nately not permanent and upon illumination these reductively bleached papers undergo rapid photoyellowing Fluorescence microscopy and microspectrofluorimetry have been used to study these and related processes 33 Of particular value was the use of sequential irradiation In this procedure the sample is irradiated with UV light (broad band pass filter centred at 365 nm) with continuous monitoring of fluorescence intensity for a set period and then it is irradiated with visible light (450-490 nm) with continu- ous monitoring of fluorescence intensity for a set period This pro- cedure is facilitated by the microscopes having the appropriate filters fitted and therefore change of excitation and monitoring wavelengths can be readily accomplished A typical result is shown in Fig 27 Initial cycle s-dSecond cycle Irradiation Blue light irradiation 30 ' 10' ' ' ' 5 ' ' ' ' tlmin Figure 27 Changes in fluorescence intensity caused by sequential UV and visible irradiation of a paper made from a high yield pulp (SGWP) Irradiation with UV light leads to a decrease in fluorescence intensity as does irradiation with visible light However following irradiation with visible light it is found that the initial intensity of the UV-stimulated fluorescence is substantially greater than that recorded at the end of the first UV excitation period Irradiation of the paper with UV light in the second cycle leads to an increase in the intensity of the fluorescence initially recorded in the second cycle of irradiation with visible light These observations are most readily accommodated by the view that lignin exhibits photochrom- ism and that the photochromic process exhibits fatigue (Fig 28) That the observed changes in fluorescence are due to lignin is attested by the fact that sections of wood when subjected to the sequential irradiation procedure exhibits the same characteristics as the paper When either the paper or wood is subjected to oxidative or reductive bleaching the intensity of the UV-stimulated fluo-rescence increases markedly Not only is this apparent when the sequential irradiation procedure is employed but also the CHEMICAL SOCIETY REVIEWS 1996 M-B M-B -Lignin mohf affectedby 365nm radiation affectedby vislbIe radiation M-V-Lignin mohf 1 p;Oducts Products Figure 28 Photochromic behaviour exhibited by lignin irreversible destruction of the chromophores giving rise to the UV- stimulated fluorescence Some differences were however observed between the behaviour of reduced wood and paper since the paper contains species which fluoresce in the visible which are not reduced by borohydride These species are thought to be stilbenes which are produced during pulping via a mechanochemical process The stilbenes are remarkably chemically active and represent an important seat of photochemical instability 33 Determination of TgValues The chemical characterisation of a crosslinked synthetic polymer is usually difficult since normally the material is insoluble in most solvents A property of some importance is its Tg value i e the temperature at which the polymer softens thereby allowing some molecular movement Techniques such as differential scanning calorimetry (DSC) and dynamic mechanical thermal analysis are frequently used but they have their limitations A method employ- ing fluorescence microscopy has been developed which allows the Tgof films including thin films and fibrous materials to be deter- mined The basis of the method lies in the technique of disperse dyeing which 1s used to dye synthetic fibres such as polyesters and polyamides In this mode of dyeing the substrate is heated in a dye-bath the temperature of which is sufficiently high as to cause the substrate to soften The dye which is present in the dye-bath in dispersed form enters the softened fibre In the attachment for the inverted fluorescence microscope shown in Fig 17 a polymer film or other substrate is laid down on the top of a few dye crys- tals It is important that the dye used is reasonably soluble in the softened polymer and that it exhibits characteristic easily visible fluorescence In our hands perylene and 3,7-bis(4-n-propoxyphenyl)benzo[ 1,2-6 4 5-b']difuran-2,6-dione have proved to be very suitable Using the attachment shown in Fig 17 the film is heated slowly When the Tg is reached the dye enters the polymer imparting a beautiful colour to the polymer (Figs 29 and 30) Tg values of several UV-cured films have been determined (Table 1 p 252) To ensure that the technique was reliable the Tgof the poly(iso- bornyl acrylate) film was also determined by DSC and was found to be very similar In another check the Tgof a commercial sample of polystyrene was determined by both techniques with the same result Given the system of heating the sample there was some concern that the heat flow from the surface nearest the heater to the surface farthest away from the heater may be so slow that accurate Tgvalues would only be obtained by ramping the temperature at an incredibly slow rate To test this point the Tgof a polyester fabric was determined both as a single and triple layer and the fact that similar results were obtained suggests that heat transfer in the system is not a problem The technique has been used to show that UV-initiated polymerisation of isobornyl acrylate produces a film having a Tgof 69 "C and that if films of isobornyl acrylate contain- ing increasing amounts of ethoxylated phenol acrylate are polymer- ised the Tgof the resulting films decreases as the amount of the latter is increased (Table 2 p 252) In many UV-initiated free radical polymensation processes the photoinitiator system consists of an aromatic ketone admixed with a tertiary amine The triplet state of the aromatic ketone reacts with the amine to give an a-aminoalkyl radical which then initiates polymensation There has been some evidence presented which supports the view that if the amines are used at a sufficiently high APPLICATIDN OF FLUORESCENCE MICROSCOPY TO A STUDY OF CHEMICAL PROBLEMS- R.S. DAVIDSON 25 1 Figure 29 Polymer film together with crystals of a benzodifuranone dye before migration of the dye has occurred. Figure 30 Polymer film together with crystals of a benzodifuranone dye showing the dye dissolving in the polymer film. concentration they can act as plasticisers. This contention is to study the migration of fluorescent species in films and across the admirably supported by the results shown in Table 3 where it can points of contact between one pdymer and another. be seen that use of ethyl 4-dimethylaminobenzoate at a concentra- Acknowledgements The work described would not have been pos- tion of >2% m/m leads to a decrease in the Tgvalues of the cured sible without the sterling efforts and intellectual input of many of films. my research students postdoctoral fellows and colleagues in indus-Undoubtedly the technique described will undergo further try. Similarly without the financial input from the SERC the developments,e.g.automation and also undergo modification so as International Wool Foundation the Australian Wool Corporation 252 CHEMICAL SOCIETY REVIEWS 1996 the EEC (contracts MAlB 0127 -C (EDB) and MA2B0018) the Table 1 Tgvalues of UV-cured films using fluorescence technique Groupemente de Recherche Papiers et DCrivCs (CNRS CTP Reactive diluents Film formed TJT Grenoble) Du Pont Demours Deutschland SmithKline Beecham Ltd and the Cancer Research Campaign it would have been impos- Isobornyl acrylate Very hard brittle film 69 sible to carry out the work Epoxidised soya bean oil Very flexible rubbery film 44 Polyester acrylate Flexible strong film 53 4 ReferencesPoly (propy lene glycol) Dye migrated at room 1 C A Parker Photoluminescence of Solutions Elsevier Amsterdam monoacrylate Very soft flexible film temperature 1968 E J Bowen Luminescence in Chemistry Van Nostrand London Ethoxylated 1968 phenol Dye migration at room 2 The Chemistry and Application of Dyes ed D R Waring and G Hallas monoacrylate Soft flexible film temperature Plenum Press New York 1990 Urethane 3 For a description of isomerisation processes exhibited by excited states oligomer/ see N J Turro Modern Molecular Photochemistry Benjamin/ poly(ethy1ene Cummins Menlo Park California 1978 glycol 200) 4 R S Davidson and M M Hilchenbach Photochem Photobiol 1990 diacrylate 75/25 Very flexible strong film 63 52,43 1 Urethane 5 B S Hudson D L Harris R D Lucheser A Ruggier A Cooney- oligomed Freed and S A Cavalier in Applications of Fluorescence in the et hoxy I ated Biomedical Sciences ed D L Taylor A S Waggoner R F Murphy F phenol Lamni and R R Birge A R Liss New York 1986 p 159 monoacry late 6 A Weller Progr React Kinet ,1%1,1,187 75/25 Very flexible strong film 40 7 G S Beddard S Carlin and R S Davidson J Chem Soc ,Perkin Urethane Trans 2,1977,262 oligomerl 8 L Tanasugarn P McNeil G Reynolds and D L Taylor J Cell Biol Tri (prop ylene 1984,98,717 glycol) diacrylate Strong film slightly 9 2 R Grabowski K Rotkiewicz A Siemarczuk J Cowley and W 75/25 flexible 60 Baumann Nouv J Chim 1979,3,443 W Rettig Angew Chem ,Int Poly (ethylene Ed Engl 1986,25971 glycol 200) 10 R S Davidson and J B Hobbs in Natural Products ed J Mann R S Davidson J B Hobbs D V Banthorpe and J V Harborne Longman diacrylate Weak flexible film 64 Poly(ethy1ene Harlow 1994 ch 3 p 131 glycol 400) 11 J C Song and D C Neckers American Chemical Society Division of diacrylate Soft flexible film 28 PMSE Papers I994,71,71 1 6-Hexanediol Brittle film slightly 12 R S Davidson In Molecular Association I ed R Foster Academic diacrylate flexible 52 Press 1975 p 215 Tn( propylene 13 G S Beddard R S Davidson and TD Whelan Chem Phys Lett glycol) diacrylate Hard film slightly flexible 59 1977,56,54 14 A P de Silva and H Q N Gunaratne J Chem Soc ,Chem Commun 1990,186 15 S A Jonker,F Ariessand J W Verhoeven Recl Trav Chim Pays-Bas 1989,108,109 S A Jonker K Van Dijk K Goubitz C A W Reiss W Schuddeboom and J W Verhoeven Mol Cryst Liq Cryst 1990 183,273 16 A P de Silva and S A de Silvo J Chem Soc ,Chem Commun ,1986 Table 2 Effect upon Tgof films produced from a mixture of 1707 E U Akkaya M E Huston and A W Czamik J Am Chem isobornyl acrylate and ethoxylated phenol acrylate Soc ,1990,112,3590 17 M Kasha J Chem Phys ,1952,20,71 Poly(isobomy1 18 R S Davidson R Bonneau J Joussot Dubien and K R Trethewey,acry1ate)lethoxylated Chem Phys Lett 1980,74,318 phenol acrylate TfC 19 R S Davidson G Ismail and D M Lewis J SOC Dyers Colourists 1oo/o 69 1988,104,86 9515 63 20 J F McKellar and N S Allen Photochemistry of Man-made Fibres 90110 58 Applied Science Publishers Barking Essex 1979 85/15 45 21 J W Longworth in Creation and Detection of the Excited State /A ed 80/20 34 A A Lamolo Marcel Dekker New York 1971 p 343 75/25 Dye migrated at room temperature 22 E S Cockburn R S Davidson and J E Pratt,J Photochem Photobiol A Chem ,1996,94,83 23 F W D Rost in Fluorescence Microscopy vol I Cambridge University Press Cambridge 1992 24 R S Davidson L Merritt and G Bradley in Aspects of Analysis Proceedings of a Conference organised by the Paint Research Association Egham Surrey 1994 25 G Bradley S Collins and R S Davidson Rev Sci lnstr ,in the press Table 3 Effect of added amine upon the Tgof cured isobornyl 26 S Collins R S Davidson P H Greaves M Healey and D M Lewis acrylate films J Soc Dyers Colourisrs 1988,104,348 27 S Collins R S Davidson and M E C Hilchenbach Dyes and N Methyldiethanolamine Ethyl 4-dimethylaminobenzoate Pigments 1994,24,15 I Amine (%) TJ0C 7'pI.C 28 K Schafer J Soc Dyers Colourists 1993,109,202 0 22 43 29 R S Davidson and Y Xue J Soc Dyers Colourists 1994,110,24 1 -53 30 S Collins and R S Davidson J Photochem Photobiol A Chem 2 31 65 1994,77,277 4 35 42 31 C Heitner in Photochemistry of Lignocellulosic Materials ACS Symp 6 38 35 Ser 53 1 ed C Heitner and J L Scaiano American Chemical Society 8 20 31 Washington DC USA 1993 p 2 A Castellan L'Actualite Chimique 10 20 30 1994 Supplement 7,148 32 R S Davidson L A Dunn A Castellan and A Nourmamode J Photochem Photobiol A Chem ,1991,58,349 APPLICATION OF FLUORESCENCE MICROSCOPY TO A STUDY OF CHEMICAL PROBLEMS-R S DAVIDSON 33 H Choudhury S Collins and R S Davidson J Photochem Photobiof H Choudhury. R S Davidson and S Greher J Photochem Photobiol A Chem 1992 69 109 A Castellan and R S Davidson A Chem 1994,81 117 A Castellan H Choudhury R S Davidson J Photochem Photobiol A Chem 1994 78,275. A Castellan and S Grelier J Photochem Photobiol A Chern 1994,81,123